PROJECT SUMMARY Preclinical and Phase II clinical data have shown that muscarinic acetylcholine receptor (mAChR) agonists, such as the M1/M4 preferring agonist xanomeline, are effective in improving both positive and negative symptoms and cognitive impairments observed in individuals with schizophrenia. However, the relative contributions of M1 and M4 mAChRs to the clinical effects of xanomeline or its effects in associated animal models remain unknown. Recently, we reported the development of a novel approach to selectively activating individual mAChR subtypes, particularly the M4 mAChR, using highly selective positive allosteric modulators (PAMs). These compounds do not activate M4 directly, but dramatically potentiate the response of the receptor to ACh. The first series of M4 PAMs, represented by VU10010, induces a 47-fold potentiation of the M4 ACh concentration response curve, possesses an EC50 in the 400 nM range, and causes no activation of the other mAChR subtypes. While VU10010 provides an important tool for proof of concept studies on the role of positive allosteric modulation of M4 at molecular and cellular levels, this compound is not suitable for in vivo studies. We have now developed several novel analogs of VU10010 that are systemically active and more readily cross the blood brain barrier. These compounds, represented by VU152100, provide an unprecedented opportunity to investigate whether the neurochemical and behavioral effects of mAChR agonists, such as xanomeline, thought to be important for antipsychotic activity and enhancement of cognition are mediated by M4. Our preliminary studies suggest that VU152100 has robust efficacy in at least one animal model used to predict antipsychotic efficacy. In the proposed studies, we will take advantage of these novel M4 PAMs along with mAChR KO mice to rigorously test the hypothesis that selective potentiation of M4 activity will have activity in animal models that predict efficacy in the treatment of schizophrenia comparable to the effects observed with xanomeline and to test the hypothesis that increased activity of M4 will regulate mesolimbic dopamine neurotransmission and transmission at glutamatergic synapses in the mPFC that are thought to be important for antipsychotic efficacy of know therapeutic agents.